TRAIL及其受体对煤工尘肺患者肺泡巨噬细胞凋亡调控作用

Regulatory role of TNF-related apoptosis-inducing ligand and its receptors in alveolar macrophage apoptosis of coal workers' pneumoconiosis

目的探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)及其受体(TRAILR)信号转导通路在煤工尘肺(CWP)患者肺泡巨噬细胞(AM)凋亡中的作用机制。方法以接触煤尘及煤矽尘的24例观察对象和CWP壹期、贰期患者各22例为研究对象,收集大容量肺灌洗回收液,分离、纯化AM后,分为对照组、超氧化物歧化酶(SOD)组、TRAIL组、抗-TRAIL抗体组、天冬氨酸特异性半胱氨酸蛋白酶(Caspase)-8抑制组、核转录因子(NF)-κB抑制剂组,给予相应处理后进行培养。收获细胞后,检测细胞凋亡情况,并提取蛋白采用免疫印迹法检测TRAIL/TRAILR信号转导通路Caspase-3、Caspase-8和死亡受体5(DR5)蛋白相对表达水平。结果 5个处理组AM凋亡指数均低于对照组(P<0.05),TRAIL组AM凋亡指数分别高于SOD组、抗-TRAIL抗体组、Caspase-8抑制剂组和NF-κB抑制剂组(P<0.05);CWP壹期和贰期患者AM凋亡指数均高于观察对象(P<0.05),CWP贰期患者AM凋亡指数高于CWP壹期患者(P<0.05);不同处理和不同程度患者AM凋亡指数的交互作用有统计学意义(P<0.01)。5个处理组Caspase-3、Caspase-8和DR5的蛋白相对表达水平分别低于对照组(P<0.05),TRAIL组Caspase-3和Caspase-8的蛋白相对表达水平分别高于SOD组、抗-TRAIL抗体组、Caspase-8抑制剂组和NF-κB抑制剂组(P<0.05),SOD组DR5蛋白相对表达水平分别低于TRAIL组、抗-TRAIL抗体组、Caspase-8抑制剂组和NF-κB抑制剂组(P<0.05);CWP壹期和贰期患者Caspase-3、Caspase-8和DR5的蛋白相对表达水平均高于观察对象(P<0.05);不同处理和不同程度患者Caspase-3的蛋白相对表达水平的交互作用有统计学意义(P<0.01),不同处理和不同程度患者Caspase-8和DR5的蛋白相对表达水平的交互作用均无统计学意义(P>0.05)。结论 TRAIL/TRAILR信号转导通路介导的AM凋亡可能在CWP发病中发挥一定作用;在不同环节对TRAIL/TRAILR信号转导通路进行抑制或阻断,可有效抑制CWP患者AM凋亡的发生。

Objective To study the mechanism of tumor necrosis factor-related apoptosis-inducing ligand（ TRAIL） and its receptors（ TRAILR） signal transduction pathways in the apoptosis of alveolar macrophage（ AM）,which were collected from coal workers＇ pneumoconiosis（ CWP）. Methods Twenty-four observed subjects exposed to coal dust and coal silica dust,22 cases of CWP at stage Ⅰ,and 22 cases of CWP at stage Ⅱ were chosen as the subjects. The bronchoalveolar lavage fluids of whole-lung lavage of all subjects were collected. AM were separated and purified. Then they were divided into 6 groups： a control group,a superoxide dismutase（ SOD） group,a TRAIL group,an anti-TRAIL antibody group,a Caspase-8 suppression group and a nuclear factor-κB（ NF-κB） suppression group. The AM of 6 groups with corresponding treatment were cultivated. After the cells were harvested,apoptosis were detected,proteins were extracted by Western blotting and relative expression levels of Caspase-3,Caspase-8 and death receptor 5（ DR5） protein in the TRAIL / TRAILR signal transduction pathway were detected. Results The apoptotic indexes of AM in 5 treatment groups were lower than that of the control group（ P〈0. 05）. The apoptotic index of AM in TRAIL group was higher than those of other groups except the control group（ P〈0. 05）. The apoptotic indexes of AM in CWP patients at stage Ⅰ and stage Ⅱ were significantly higher than those of the observed subjects respectively（ P〈0. 05）. The apoptotic indexe of AM in CWP patients at stageⅡ was significantly higher than that of CWP patients at stageⅠ（ P〈0. 05）. The interaction of different treatment and different degree patients had significant difference in apoptotic indexe of AM（ P〈0. 01）. The protein relative expression levels of Caspase-3,Caspase-8 and DR5 in 5 treatment groups were significantly lower than those of the control group respectively（ P〈0. 05）. The protein relative expression levels of Caspase-3 and Caspase-8 in TRAIL group were significantly higher than those of other groups except the control group（ P〈0. 05）. The protein relative expression levels of DR5 in SOD group were significantly lower than those of other groups except the control group（ P〈0. 05）. The protein relative expression levels of Caspase-3,Caspase-8 and DR5 in CWP patients at stage Ⅰ and stage Ⅱ were significantly higher than those of the observed subjects（ P〈0. 05）; the relative expression levels of protein Caspase-3 in different treatments and different degree patients had statistical significant difference in interaction（ P〈0. 01）,while the protein relative expression levels of Caspase-8 and DR5 in different treatments and different degree patients had no statistical significant difference in interaction（ P〈0. 05）. Conclusion AM apoptosis mediated by TRAIL / TRAILR signal transduction pathway may play a certain role in the occurrence and development of CWP. The TRAIL / TRAILR signal transduction pathways inhibited or blocked at different stages can effectively inhibit the occurrence of AM apoptosis.